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dCas9-KRAB CRISPRi(CRISPR interference)慢病毒载体系统

概述

Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR Associated Protein 9(CRISPR/Cas9)系统是近年新兴的基因编辑工具之一,另外两种是ZFN和TALEN。由于CRISPR/Cas9的构建更简易,超越了更早开发的ZFN和TALEN技术,成为现今基因编辑领域最炙手可热的技术。CRISPR/Cas9技术源于原核免疫系统CRISPR/Cas。在自然界中,细菌利用CRISPR/Cas抵抗质粒和噬菌体等外源遗传物质的入侵,从而保持自身基因组的完整性。

两个生物大分子,Cas9蛋白和gRNA(guide RNA)组成了CRISPR/Cas9基因编辑系统。在细胞内,Cas9蛋白与gRNA形成复合物,能特异性鉴别出靶序列。此过程中,Cas9蛋白负责将复合物定点到靶DNA和剪切靶DNA。Cas9蛋白有6个结构域,分别是Rec I、Rec II、Bridge Helix、PAM Interacting、HNH和RuvC。Rec I是6个中最大的一个结构域,负责结合gRNA。一旦结合了靶DNA,Bridge Helix就负责启动剪切。PAM interacting结构域赋予Cas9对PAM序列的特异性要求,负责启动与靶DNA的结合。HNH和RuvC都是核酸酶结构域,剪切单链DNA。

图1. Cas9蛋白结构域 

在没有结合gRNA的情况下,Cas9蛋白保持失活状态。gRNA是一条单链RNA,会形成一个T型结构。在这个T结构里,有几个显著的特征:tetraloop、3个stem loop和5’ 端与靶DNA互补的序列。

图2. gRNA结构

一旦gRNA结合上Cas9蛋白,就会引起Cas9蛋白的构象改变。这种构象的改变使得Cas9蛋白从失活状态变成了活性状态。

图3. Cas9构象改变

一旦Cas9蛋白被激活,它就开始寻找靶DNA。此过程中,它首先结合到含有PAM(protospacer adjacent motif)序列的序列。一段PAM序列就是2个或者3个碱基序列,位于与gRNA互补的靶DNA序列的下游,紧跟着靶DNA序列。不同的CRISPR系统含有不同的PAM序列,例如:广泛应用的Streptococcus pyogenes Cas9的PAM序列是5′-NGG-3′。Jiang等人证明SpCas9也可以识别NAG,但是效率较低。当Cas9结合到含有PAM序列的潜在靶DNA序列,它会解开PAM的上游双链DNA序列。此时,gRNA的5’ 端序列与解开的序列配对。一旦配对成功,RuvC和HNH核酸酶结构域就会在PAM序列上游的第3个碱基和第4个碱基之间切开。RuvC剪切下链,HNH剪切上链。

图4. Cas9结合并剪切靶DNA

CRISPRi技术依靠生成一个没有核酸酶活性的Cas9蛋白。这是通过往RuvC和NHN两个核酸酶结构域分别导入氨基酸突变D10A和H840A,使得Cas9蛋白失去切割DNA活性,但仍保留结合DNA的能力,这样的Cas9 称之为dCas9(Dead Cas9)。

图5. dCas9结构域

当dCas9被引导到某个基因的转录起始位点TSS(transcription start site)时,dCas9能够物理性阻碍RNA聚合酶的通过,导致基因沉默。为了进一步提高转录抑制的效率,dCas9融合了一个基因抑制结构域,如KRAB(krüppel-associated box)结构域,这样的蛋白称之为dCas9-KRAB。此外,dCas9也可以融合双抑制结构域(bipartite repressor domain)KRAB-MeCP2,抑制效果更佳。

图6. dCas9-KRAB CRISPRi工作原理

一个完整的dCas9-KRAB CRISPRi慢病毒载体系统包含两个部分,gRNA表达载体和dCas9-KRAB(或者dCas9-KRAB-MeCP2)表达载体。设计基因抑制实验时,只需要设计打靶目的基因的gRNA表达载体即可。

图7. dCas9-KRAB CRISPRi慢病毒载体系统

关于该载体系统的更多信息,请参考以下文献:

参考文献 主题
Cell. 154:442 (2013) Characterization of CRISPRa and CRISPRi systems
Nat Methods. 12:1143 (2015) Characterization of the dCas9-KRAB system
Nat Methods. 15:611 (2018) Characterization of the dCas9-KRAB-MeCP2 system
优势

不改变内源基因组背景:与CRISPR基因编辑、传统基因敲除技术不同,dCas9-KRAB CRISPRi系统不会改变靶基因位点基因组序列。

强基因抑制效果:使用dCas9-KRAB CRISPRi系统进行转录抑制通常可以获得高水平的基因抑制效果。

更多适用的基因种类:由于dCas9-KRAB CRISPRi系统是在DNA水平抑制基因表达,因此适用于多种转录本,包括mRNA、非编码RNA、microRNA、反义转录本、核定位RNA以及聚合酶III 转录本的转录抑制。

特异性:dCas9-KRAB CRISPRi系统可实现高效抑制同时,几乎没有脱靶现象。

不足之处

不同基因之间差异性:由于dCas9-KRAB需要接触到目的基因的调控序列,因此会因为基因所处染色体位置不同而产生不同的抑制程度,这取决于它们的内源染色质状态。

载体元件

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of 5'LTR-ΔU3 LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

U6 promoter: This drives high level expression of the downstream user-selected gRNA.

gRNA: Allows in vitro transcription for RNA preparation. Scaffold gRNA sequence is included.

Terminator: Terminates transcription of the gRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.